Model Introduction
Fungal Keratitis (FK) is an infectious corneal disease with a high rate of blindness, which can lead to eyeball loss in severe cases. As an agricultural nation, China has maintained a high incidence of FK, with an upward trend in recent years. Its occurrence is closely related to the widespread use of broad-spectrum antibiotics and glucocorticoids, long-term contact lens wear, and the use of immunosuppressants. Due to the vast variety of pathogenic fungi and complex infection mechanisms, research on the pathogenesis of corneal fungal infection remains limited, and clinical anti-fungal drugs often suffer from high toxicity or low efficacy. Therefore, establishing stable and reliable animal models is essential for studying the pathogenesis and therapeutic strategies of FK. Currently, there is a lack of ideal animal models that can perfectly simulate the human corneal fungal infection process, which to some extent restricts the development of basic research.
Research Applications
Animal models of fungal keratitis are primarily used for:
- Researching the pathogenesis of corneal fungal infection.
- Analyzing the histopathological changes of corneal infection.
- Evaluating the efficacy of anti-fungal drugs.
- Studying corneal immune responses and inflammatory mechanisms.
- Developing new diagnostic and therapeutic methods. Research typically involves clinical manifestations, microbiological characteristics, histopathological changes, and molecular mechanisms.
Key Points of Experimental Design
I. Selection of Experimental Animals
Animals commonly used in fungal infection research include:
- Mice
- Guinea pigs
- Hamsters
- Rabbits
- Birds
- Dogs
- Cats Among these, mice and rabbits are the most widely used.
Mouse Model Characteristics Pros:
- Low cost.
- Easy to house.
- Suitable for large-scale experiments. Cons:
- Small cornea.
- Difficult surgical operation.
- Not conducive to gross observation.
Rabbit Model Characteristics Rabbits are currently the most commonly used animals for studying corneal fungal infections. Pros:
- Large cornea.
- Convenient surgical operation.
- Easy local observation.
- Allows for obtaining more experimental specimens. Common Strains:
- New Zealand White Rabbit.
- Dutch Belted Rabbit.
II. Selection of Pathogenic Fungal Strains
Corneal infectious fungi are mainly divided into two categories: 1. Filamentous Fungi
- Fusarium spp.
- Aspergillus spp.
- Penicillium spp.
- Dematiaceous fungi 2. Yeasts
- Candida spp.
Pathogenic strains vary by region:
- Developed countries and cold regions: Candida albicans is common.
- Developing countries and warm regions: Fusarium and Aspergillus are more common. Survey results in China show the main pathogens are:
- Fusarium (most common).
- Aspergillus.
In experimental research, the following are commonly used:
- Fusarium
- Aspergillus
- Candida albicans To ensure reliability, it is recommended to use:
- ATCC standard strains.
- Strains preserved by the Institute of Microbiology, Chinese Academy of Sciences.
- Clinical isolates from patients.
III. Fungal Inoculation Methods
1. Preparation of Fungal Suspension The concentration is usually: 10⁶–10¹⁰ CFU/ml. To ensure infection success, the concentration typically needs to be ≥10⁷ CFU/ml. Common inoculation volume: 0.005–0.1 ml.
2. Corneal Inoculation Methods (1) Intrastromal Injection Method Operation:
- Use an insulin syringe with a 28 or 30-gauge needle.
- Inject approx. 0.05 ml of fungal suspension.
- Inject into the central 1/3 depth of the corneal stroma. In some experiments, glucocorticoids (e.g., Triamcinolone acetonide) are injected subconjunctivally the day before inoculation. Characteristics: Pros: Simple operation; suitable for drug efficacy studies. Cons: Alters the natural infection process; disrupts corneal structure; unsuitable for pathogenesis studies.
(2) Contact Lens Method Steps:
- Scrape the central 7 mm area of the rabbit corneal epithelium.
- Cover with a soft contact lens.
- Inject fungal suspension between the lens and the cornea.
- Perform temporary tarsorrhaphy.
- Remove the contact lens after 24 hours. Characteristics: Pros: Approaches the natural infection process; high infection success rate. Cons: Usually requires high-concentration suspension; disease progression may be more severe than clinical cases.
(3) Corneal Overlay (Keratoprosthesis) Method Operation:
- Scrape approx. 6 mm of the central corneal epithelium.
- Suture a full-thickness allogeneic corneal graft onto the corneal surface.
- Inject fungal suspension into the interlaminar space.
- Remove the graft after 24 hours. Characteristics: Pros: Does not depend on glucocorticoids; uses relatively low fungal concentrations; infection success rate near 100%; good reproducibility. Cons: Complex surgical steps; requires high technical skill.
Key Detection Indicators
I. Identification of Fungal Infection Fungal Culture (Standard Method) Medium:
- Sabouraud Dextrose Agar (SDA).
- Supplemented with 0.05% chloramphenicol. Incubation Time: ≥72 hours. Identification:
- Molds: Morphological identification.
- Yeasts: Morphological + physiological identification. Fungal culture is the Gold Standard for diagnosis.
II. Histopathological Examination Histological analysis of the infected cornea:
- Corneal inflammatory response.
- Distribution of fungal hyphae.
- Degree of tissue damage. Immunohistochemical (IHC) detection:
- Fungal-specific antigens (higher sensitivity than traditional staining).
III. Corneal Lesion Scoring Observe corneal infection via slit-lamp microscopy and perform clinical scoring. Scoring standards (Modified O’Day Method) include:
- Area of corneal infection (Grade 0–4).
- Depth of corneal lesion (Grade 0–4).
- Anterior chamber inflammatory response (Grade 0–4). The total of these three scores provides a composite score to quantify infection severity.
